Diphosphopyridine Nucleotide by Ricinus Mitochondria

Transcription

1 Vol. 76 EFFECTS OF C02 ON MITOCHONDRIAL ENZYMES 221 Foster, W. E. (1940). Ph.D. Thesis: Durham University. Harrison, A. (1953). Ph.D. Thesis: Durham University. Hulme, A. C. (1956). Nature, Lond., 178, 218. Leonard, 0. A. (1945). J. Amer. Soc. Agron. 37, 55. Magness, J. R. (1920). Bot. Gaz. 70, 308. Ranson, S. L. (1953). Nature, Lond., 172, 252. Ranson, S. L. (1955). In Modern Methods of Plant Analysis, vol. 2, p Ed. by Paech, K. & Tracey, M. V. Berlin: Springer Verlag. Ranson, S. L., Walker, D. A. & Clarke, I. D. (1957). Biochem. J. 66, 57P. Russell, E. J. & Appleyard, A. (1915). J. agric. Sci. 7, 1. Small, J. (1954). Modern Aspects of ph, chapt. 6. London: Bailli6re, Tindall and Cox. Smith, W. H. (1957). Nature, Lond., 179, 876. Thomas, M. (1925). Biochem. J. 19, 927. Thornton, N. C. (1933). Contr. Boyce Thompson Inst. 5,403. Umbreit, W. W., Burris, R. H. & Stauffer, J. F. (1949). Manometric Techniques and Tissue Metabolism, chapt. 3. Minneapolis: Burgess Publishing Co. Ulrich, R. & Landry, J. (1956). C.R. Acad. Sci., Paris, 242, Walker, D. A. & Beevers, H. (1956). Biochem. J. 62, 120. Walker, D.A. & Brown, J. M. A. (1957). Biochem. J. 67, 79. Biochem. J. (1960) 76, 221 Effects of Carbon Dioxide on the Oxidation of Succinate and Reduced Diphosphopyridine Nucleotide by Ricinus Mitochondria BY D. S. BENDALL Department of BiocheMi8try, Univer8ity of Cambridge S. L. RANSON Department of Botany, King'8 College, Newca8tle upon Tyne AND D. A. WALKER Department of Botany, Queen Mary College, London, E. 1 (Received 29 December 1959) The oxidation of succinate to fumarate by particulate preparations from Ricinu8 endosperm is retarded in atmospheres containing more than about 10% of carbon dioxide (Ranson, Walker & Clarke, 1957, 1960) and the retardation has been attributed to an inhibition of the succinic-oxidase system of the mitochondria. The present paper is an enlargement of a preliminary account (Bendall, Ranson & Walker, 1958) and describes the results of an investigation to determine which components of the respiratory chain are inhibited. The effects of carbon dioxide-bicarbonate mixtures on the succinic-cytochrome c reductase system and on cvtochrome c oxidase were determined, and evidence is presented that the actual site of inhibition is succinic dehydrogenase. Since previous work had also suggested that the enzymic reoxidation of reduced diphosphopyridine nucleotide may be affected by high concentrations of carbon dioxide, the effects of carbon dioxidebicarbonate mixtures on the reduced diphosphopyridine nucleotide-cytochrome c reductase system were also determined. In an attempt to assess the effects of the sodium bicarbonate in the mixtures with carbon dioxide the effects of some other salts have also been studied. MATERIALS AND METHODS Cytochrome c was purchased from the Sigma Chemical Co., St Louis, Mo., U.S.A. and Boehringer und Soehne, Mannheim, Germany. It was dissolved in 1% (w/v) 2- amino-2-hydroxymethylpropane-1:3-diol (tris)-hcl buffer, ph 7-4, and estimated spectrophotometrically at 550 m,u. Molar extinction coefficients of 27-7 x moles-l cm.-i for the reduced form and 9-2 x moles-1 cm.-" for the oxidized form were used (Margoliash, 1954). Reduced cytochrome c was prepared by adding a slight excess of sodium dithionite and bubbling 02 through the solution for 5 min. to remove the excess of dithionite. Reduced diphosphopyridine nucleotide (DPNH) was purchased from the Sigma Chemical Co. Preparations of mitochondrial fractions. Suspensions of mitochondria from the endosperm of castor beans (Ricinus communis var. Cimarron) were prepared as described previously (Ranson et al. 1960). 30 min. before spectrophotometer experiments the suspensions were diluted tenfold with ice-cold demineralized water, since it has been found that Ricinus mitochondria will not react easily with added cytochrome c when suspended in 0 5M-sucrose but react readily after incubation in 0 05 M-sucrose. This is attributed to the osmotic rupture of membranes which would otherwise limit the free access of cytochrome c to enzymic centres. Enzyme assays. Succinic-cytochrome c reductase, DPNH-cytochrome c reductase and cytochrome c oxidase activities were measured spectrophotometrically by

2 222 D. S. BENDALL, S. L. RANSON AND D. A. WALKER 1960 methods already described (Bendall et al. 1958) with a mitochondria, with succinate as substrate, was Unicam SP. 500 spectrophotometer in a temperaturecontrolled room at 250. dioxide-bicarbonate mixture markedly inhibited in the presence of carbon Spectro8copic ob8ervations. at a final These were made with concentration a Zeiss microspectroscope ocular. The scale was set with the a-band corresponding to 30% of carbon dioxide. No of reduced cytochrome c at 550 mp. change was detectable with the microspectroscope Regulation of carbon dioxide concentration. Conditions in the aqueous reaction mixtures corresponding to various atmospheric C00 concentrations were simulated by. the addition of C0,-bicarbonate mixtures as described (Ranson et al. 1960). The procedure was modified slightly for the spectrophotometer experiments in that the C00 concentration was regulated simply by the addition of the appropriate quantity of 0-316M-NaHC03 which had previously been brought to equilibrium at ph 7-4 and 250 with a gas mixture containing 90% of C00 and 10% of 02. The bicarbonate was added to the other components in closed spectrophotometer cells immediately before the reactions were started. Subsequent determinations of initial rates were completed within 2 min. Previous experience (Walker & Brown, 1956) had indicated that the loss of C02 from solution in these circumstances is extremely small. RESULTS Preliminary observation-s The effects of a carbon dioxide-bicarbonate mixture on the succinic-oxidase system were examined spectroscopically as follows. To a 1 cm. cell containing 1-6 ml. of a suspension of washed mitochondria was added 0-4 ml. of 0-34M-sodium bicarbonate saturated with carbon dioxide to give a final concentration corresponding to approximately 20% of carbon dioxide. A control cell was set up in which 0-34M-sodium chloride replaced the bicarbonate solution. The cells were shaken with air and to each was added 0-5 ml. of 1-2m-potassium succinate. In the control mixture the a-bands of reduced cytochromes a, b and c rapidly became visible at 603 ma, m,u and mp respectively. The a-bands of cytochromes a and c appeared almost simultaneously closely followed by the a-band of cytochrome b. The complete reduction took about 15 sec. In the cell containing bicarbonate the order of reduction of the cytochromes was the same, but the rate was slower with complete reduction being attained in about 30 sec. In both cases the cytochromes were reoxidized on shaking in air. The addition of a further 0-4 ml. of carbon dioxide-bicarbonate mixture increased the time for complete reduction to 45 sec., whereas the addition of a corresponding amount of sodium chloride to the control was without effect. Again in both cases the cytochromes were reoxidized on shaking with air. The suggestion from these observations that the inhibition by bicarbonate or carbon dioxide occurred at a point below cytochrome b in the respiratory chain was consistent with the following qualitative observations. In an evacuated Thunberg tube the reduction of methylene blue by the in the spectrum of reduced cytochrome c in the presence of a high concentration of the carbon dioxide-bicarbonate mixture. There was no detectable change in the appearance of the a-band of cytochrome a when, in an evacuated Thunberg tube, sodium bicarbonate was tipped from the side arm into a reaction mixture containing mitochondria and succinate, to yield a final concentration of 0-6M. Spectrophotometric measurement8 The effects of various concentrations of bicarbonate-carbon dioxide buffer on the succinicand DPNH-cytochrome c reductase systems and on cytochrome c oxidase are shown' in Figs The activity of succinic-cytochrome c reductase fell off rapidly with increasing carbon dioxide concentration above 5 %, as little as 12% of carbon dioxide causing a 50% inhibition. DPNH-cytochrome c reductase was also inhibited by carbon dioxide concentrations above about 30% but the activity fell offless rapidly than that ofsuccinic-cytochrome c reductase and appreciable inhibition occurred only at relatively high carbon dioxide concentrations. The carbon dioxide concentration required for 50% inhibition was about 60 %. On the other hand, cytochrome c oxidase showed a maxked increase in activity at carbon dioxide concentrations._:._ v a, 0 v c u a. Concn. of NaCI (m) Concn. of CO2 (% of I atm.) Fig. 1. Effect of C00-bicarbonate mixtures (0) and of NaCl (0) on the succinic-cytochrome c reductase system. The reaction mixtures contained, in a final volume of 3 ml.: 0-67% of tris-hcl buffer, ph 7-4, 31,M-cytochrome c, 0-5 mm-kcn, ph 7-4, 20 mm-sodium succinate, enzyme and the appropriate quantity either of 0-316M-NaHCO3, which had been equilibrated against CO (90: 10) at 25, or of 0-316mr-NaCl. Temp. 25.

3 Vol. 76 EFFECTS OF C02 ON SUCCINATE AND DPNH OXIDATION 223 and 5 the results have been plotted according to the method of Lineweaver & Burk (1934) for the succinic- and DPNH-cytochrome c reductases around 20% but at higher concentrations the activity declined and above 60% of carbon dioxide the activity was markedly below that in air. The degree of stimulation observed in different preparations was rather variable, and in one case the maximum activity was double that in the absence of carbon dioxide. In order to obtain further information about the types of inhibition involved with the cyto hrome reductases the rates of reaction were measured at different concentrations of succinate or DPNH for a series of carbon dioxide concentrations. In Figs. 4 respectively. It seems clear that the inhibition of succinic-cytochrome c reductase was essentially of the competitive type, whereas that of DPNHcytochrome c reductase was non-competitive. However, the Lineweaver-Burk plots for the succinic system passed through a point which lay a little off the 1/v axis, which suggests that there may have been a small contribution from a non- Concn. of NaCI (M) (0( _ )._._! C._i U Es 0 4, u L. Concn. of CO2 (% of 1 atm.) Fig. 2. Effect of 00,-bicarbonate mixtures (0) and of NaCl (0) on the DPNH-cytochrome c reductase system. The reaction mixtures were as given in Fig. 1 except that sodium succinate was replaced by mm-dpnh. Temp /v /s Fig. 4. Effect of succinate concentration on succiniccytochrome c reductase at different concentrations of CO,- bicarbonate buffer. v, reaction velocity (pnmoles of cytochrome c/min./g. of fresh tissue); 8, succinate concn. (M). 0, 17-3% of CO,; *,8-7% of CO,; A, 4.3 % of CO,; x, no CO. Other reaction conditions were as given in Fig. 1. Concn. of NaCI (M) } Concn. of CO2 (% of I atm.) Fig. 3. Effect of CO-bicarbonate mixtures (0) and of NaCl (0) on cytochrome oxidase. The reaction mixtures contained, in a final volume of 3 ml.: 0-67 % of tris-hcl buffer, ph 7*4, 14-9 psm-reduced cytochrome c and the appropriate quantity of either CO,-bicarbonate mixture or NaCl as in Fig. 1. Temp /S Fig. 5. Effect of DPNH concentration on DPNH-cytochrome c reductase at different concentrations of CO,- bicarbonate buffer. v, reaction velocity (,umoles of cytochrome c/min./g. of fresh tissue); S, DPNH ooncn. (mm). 0, 78% of CO.;, 60-6 % of C02; A, 43-4% of C00; x, no CO. Other reaction conditions were as given in Fig. 2.

4 224 D. S. BENDALL, S. L. RANSON AND D. A. WALKER 1960 competitive type of inhibition (Dixon & Webb, 1958). This could have been due to a non-specific salt effect of the sodium bicarbonate since sodium chloride also gave some inhibition at low concentrations (Fig. 1). The competitive nature of the inhibition of the succinic system indicates that the inhibition is reversible. The following observations suggest that the inhibitions of the other enzymic systems by high concentrations of carbon dioxide and bicarbonate are also reversible. Two samples of the original mitochondrial preparation were diluted tenfold, one by the addition of water, the other by the addition of carbon dioxide-bicarbonate buffer to give a final mixture in equilibrium with 80 % of carbon dioxide. They were allowed to stand in the cold for 30 min. Cytochrome oxidase and DPNH-cytochrome c reductase activities were then assayed in reaction mixtures containing 0-1 ml. of the treated mitochondrial preparations in a final volume of 3 ml. If the combination of carbon dioxide or bicarbonate with the enzymes had been irreversible the activities in the preparations treated with carbon dioxide-bicarbonate would have been very markedly inhibited. In fact the activities ofdpnhcytochrome c reductase and of cytochrome c oxidase in the preparation treated with carbon dioxidebicarbonate mixture were 99 and 114 % respectively of those in the sample diluted with water. Effects of 8alts other than bicarbonate To test the specificity of the inhibitions by carbon dioxide-bicarbonate mixtures, the effects of salts other than sodium bicarbonate on the three enzymic systems were investigated. The effects of sodium chloride on the DPNH-cytochrome c reductase and on the cytochrome oxidase were clearly similar to those of sodium bicarbonate (Figs. 2, 3). Inhibitory effects were obtained also with other salts, e.g. the percentage inhibitions of the reductase by sodium bicarbonate, sodium chloride, potassium chloride and sodium sulphate (each 0-274m) and by 0-137M-sodium sulphate were 56, 67, 63, 78 and 59 % respectively. In contrast the inhibition of the succinic-cytochrome c reductase by sodium chloride was markedly less than by bicarbonate (Fig. 1). The comparisons of the effects of sodium chloride and sodium bicarbonate-carbon dioxide mixture shown in Figs. 1, 2 and 3 are for equivalent concentrations of sodium ion. DISCUSSION The inhibitions of the succinic and DPNH oxidase systems by carbon dioxide-bicarbonate mixtures appear to be of two types. The first kind of inhibition affected the succinic-cytochrome c reductase system only, and it was appreciable at concentrations of carbon dioxide below 20 %. The fact that the inhibition was competitive with respect to succinate and that sodium chloride had only a small effect on the system in the equivalent concentration range both indicate that this type of inhibition is a specific effect of the carbon dioxide mixture. The competitive nature of the inhibition also suggests that the actual component of the system affected was succinic dehydrogenase. This interpretation is consistent with the observations in the spectroscope and Thunberg experiments which indicated that the inhibition occurs at some point below cytochrome b in the succinic-oxidase chain. They provided no evidence of an inhibition at any point in the chain between cytochrome b and cytochrome a. Whether the inhibition was due to carbon dioxide or bicarbonate or possibly to both components of the mixture is at present unknown. The second kind of inhibition is a relatively nonspecific effect of high salt concentration which affected all three enzyme systems studied. The similarity of the effects of sodium chloride and carbon dioxide-bicarbonate mixtures on the DPNH-cytochrome c reductase and cytochrome c oxidase systems suggests that the effects of the solutions containing high carbon dioxide concentrations on those systems was probably due not to carbon dioxide itself but to the high sodium bicarbonate concentration. It is not yet clear whether this was an effect due to the anion or the cation. The specific inhibition of the cytochrome oxidase of root mitochondria by bicarbonate observed by Miller & Evans (1956) was not observed under the different experimental conditions of the present work. From the foregoing it appears that the marked inhibition of succinic oxidase in the intact mitochondria (Ranson et al. 1960) in bicarbonate mixtures in equilibrium with carbon dioxide concentrations above about 10% can be attributed very largely to inhibition of the succinic-dehydrogenase component. The further inference from the earlier experiments that pyruvic oxidase may be inhibited at still higher concentrations of carbon dioxide and bicarbonate may possibly relate to the inhibition of DPNH-cytochrome c reductase and cytochrome c oxidase by the bicarbonate. To what extent all of these results are relevant to the effects observed when intact plant tissues are exposed to atmospheres containing high concentrations of carbon dioxide is at present uncertain. It seems probable, however, that the accumulation of succinate noted in a variety of plant tissues after storage in atmospheres containing 10-20% of carbon dioxide (Ranson, 1953; Hulme, 1956; Ulrich & Landry, 1956) is related to an inhibition of succinic dehydrogenase in the tissues. The observation that the inhibition of this enzyme by carbon

5 Vol. 76 EFFECTS OF C02 ON SUCCINATE AND DPNH OXIDATION 225 dioxide is of a competitive nature would account concentrations and inhibited by high concentrations of sodium bicarbonate and other salts. for such variation as occurs in the level of carbon dioxide required to bring about succinate accumulation in different plant tissues. the observed effects of carbon dioxide on the oxid- 5. The results have been discussed in relation to Whether or not very high concentrations of ation of succinate and other acids by intact mitochondria and the effects of carbon dioxide in the carbon dioxide in the atmosphere around a plant tissue could in fact result in a sufficiently high bicarbonate concentration in the cytoplasm to bring The work described in this paper was started and carried environment around intact plant tissues. about the inhibition of DPNH-cytochrome c out for the most part in the Department of Botany, King's reductase and cytochrome c oxidase is not known. College, Newcastle. The authors wish to express their thanks If this did occur the combined inhibition of these to Professor M. Thomas, F.R.S., for his continued interest enzymes together with that of the succinic dehydrogenase would go a long way towards explaining the with apparatus financed by the D.S.I.R. and University of and assistance. Several experiments were also carried out onset of carbon dioxide zymasis (Thomas, 1925) in London, Central Research Fund. We are also grateful for a atmospheres containing carbon dioxide in concentrations approaching 50 quantity of Ricinus seeds provided by the Baker Castor Oil Co., San % and Diego, Calif., U.S.A. One of us (D. S. B.) is indebted abundant to the Agricultural Research Council for a research studentship held while part of the work was in progress. oxygen. SUMMARY 1. The rates of three reactions concerned in the oxidation of succinate and reduced diphosphopyridine nucleotide by Ricinuw mitochondria were determined in reaction mixtures in equilibrium with various concentrations of carbon dioxide at ph 7-4 and Succinic-cytochrome c reductase was markedly inhibited by relatively low concentrations of carbon dioxide-bicarbonate but only slightly by corresponding concentrations of sodium chloride. The inhibition was competitive with respect to succinate concentration. For this and other reasons it is inferred that the carbon dioxide-sensitive oomponent of the reductase is succinic dehydrogenase. 3. Reduced diphosphopyridine nucleotide-cytochrome c reductase was inhibited by high concentrations of sodium bicarbonate and other salts. 4. Cytochrome-c oxidase was stimulated by low REFERENCES Bendall, D. S., Ranson, S. L. & Walker, D. A. (1958). Nature, Lond., 181, 133. Dixon, M. & Webb, E. C. (1958). Enzyme8. London: Longmans Green and Co. Hulme, A. C. (1956). Nature, Lond., 178, 218. Lineweaver, H. & Burk, D. (1934). J. Amer. chem. Soc. 56, 658. Margoliash, E. (1954). Biochem. J. 56, 259. Miller, G. W. & Evans, H. J. (1956). Nature, Lond., 178, 974. Ranson, S. L. (1953). Nature, Lond., 172, 252. Ranson, S. L., Walker, D. A. & Clarke, I. D. (1957). Biochem. J. 66, 57P. Ranson, S. L., Walker, D. A. & Clarke, I. D. (1960). Biochem. J. 76, 216. Thomas, M. (1925). Biochem. J. 19, 927. Ulrich, R. & Landry, J. (1956). C.R. Acad. Sci., Paris, 242, Walker, D. A. & Brown, J. M. A. (1956). Biochem. J. 67, 79. Biochem. J. (1960) 76, 225 The Measurement of the Diffusion of Oxygen through Respiring Tissue BY I. S. LONGMUIR AND ANN BOURKE Department of Biochemi8try, Institute of Diseases of the Chest, London, S.W. 3 The diffusion coefficient of oxygen through nonrespiring tissue has been measured by Krogh (1919) The figure he obtained was a little lower than that through water. Similar values have been obtained by other workers (Kirk & Johnsen, 1951). In general the diffusion coefficient of oxygen through tissue appeared to be equal to that through water multiplied by the fraction of the tissue composed of (Received 8 January 1960) water. It seemed that the transport of oxygen through tissues consisted of simple diffusion through the aqueous part uninfluenced by the solid part. That this is not universally true was shown by Longmuir & Roughton (1952), who found that the diffusion coefficient of nitrogen and carbon monoxide through haemoglobin solutions of the concentration found in red cells was about one- 15 Bioch. 1960, 76